This invention relates to an improved membrane for an enzyme electrode, and more particularly to a laminated membrane wherein the enzyme itself (with or without other materials blended with it) is used as the adhesive between the lamina.
Polarographic cell systems have become quite popular in the medical field for measurement of various substances. In addition, enzymes have been used in conjunction with polarographic cells, especially in instances where the unknown substance to be measured is not polarographically active, but a material produced or consumed by an enzymatic reaction with that unknown is detectable. For example, it is known that glucose is not polarographically active but that the following reaction takes place in the presence of the enzyme glucose oxidase: ##EQU1## The existence of this reaction is significant in enabling polarographic measurement of glucose.
Thus, in an article by Clark and Lyons in the Annals of the New York Academy of Science, 102, 29-45 (1962), it was suggested that a pH sensitive electrode could be used to detect the gluconic acid produced by the reaction. It was disclosed that the enzyme in such a system could be trapped between Cuprophane membranes. The glucose diffuses through the membrane and is converted by the enzyme to gluconic acid, which then diffuses both toward the pH sensitive glass and back into the donor solution.
Alternatively, it was suggested that by using a hydrophobic membrane, a dialysis membrane, glucose oxidase, and a pO.sub.2 electrode, a system could be arranged that is sensitive to glucose by virtue of the fact that oxygen is consumed from the flowing glucose solution in proportion to its glucose content.
Later, Clark obtained a patent on an improvement in such a system. In U.S. Pat. No. 3,539,455, it is stated that the system disclosed therein "differs in simplicity, reliability and in function from the cell disclosed in `Annals of the New York Academy of Sciences`". Rather than measuring the pH change or the oxygen consumption, the Clark patent discloses using a platinum anode to measure the hydrogen peroxide produced. In the polarographic cell described in that patent, the enzyme is placed on the anode side of a cellophane membrane. The low molecular weight glucose passes through the membrane and reacts with the enzyme, but interfering high molecular weight catalase and peroxidase materials do not. It is disclosed that the enzymes may be held in a thin film directly between the platinum surface and the membrane by placing the enzyme on a porous film which has spaces large enough to hold enzyme molecules. The use of polymeric gels to stabilize the enzyme is also disclosed.
Since the cellophane membrane will not prevent low molecular weight interfering materials such as uric acid or ascorbic acid from reaching the anode, Clark suggests a dual electrode system. The compensating electrode, without an enzyme present, gives a signal for the interfering substances while the enzyme electrode detects both the hydrogen peroxide and the interference. By subtracting the reading of the compensating electrode from that of the glucose electrode, the amount of hydrogen peroxide production, and thus, the glucose level is determined. Still, such a dual sensor system may encounter difficulties in the matching of the two cells.
Under the circumstances, then, it would be desirable to have an enzyme electrode which employs a thin filter membrane to prevent passage of even low molecular weight interfering materials, such as uric acid and ascorbic acid, while permitting hydrogen peroxide to pass therethrough with minimum hindrance. There exist membrane materials, such as silicone rubber and cellulose acetate, which permit passage of hydrogen peroxide but which are effective barriers to interfering substances. Since this type of membrane must be placed between the anode and some component of the sensing system, it follows that in order for measurement equilibrium to be as rapid as possible, the membrane must be as thin as possible while still retaining its selectivity. In the case of a hydrogen peroxide sensing probe, this membrane will need to be less than 2 microns thick. A membrane of this thickness is difficult, if not impossible to use in practice because of its insufficient strength.
Some support is needed. Depositing the material in a thin layer on a porous substructure will be in some respects satisfactory. The porous substructure will provide the necessary strength while at the same time being of little hindrance to hydrogen peroxide passage, and the weak interference rejecting layer can be thin to enhance speed of response. It remains that this laminated membrane be combined with a polarographic electrode and appropriate enzyme in such a fashion that the completed sensor responds satisfactorily to the desired non-polarographic substrate. In a common configuration with a typical membrane, the enzyme is placed between the anode and membrane as disclosed in the Clark patent. With the laminated membrane just described, the enzyme in this configuration would be as effectively shielded as the anode. Therefore the interference rejection must be limited to molecules the same size or larger than the substrate of the enzyme. Membrane materials that would reject smaller interferences would also prevent the substrate from reaching the enzyme.
Alternatively, the enzyme may be placed on the side of this laminated membrane away from the anode. In this case it may be captured by a third outer membrane layer which is permeable to the substrate but impermeable to the enzyme. In this configuration, the substrate is not unnecessarily hindered from reaction with the enzyme, and good interference rejection is possible since the filter layer need pass only the resultant polarographic substance, i.e., hydrogen peroxide. The polarographic substance, however, is now produced two layers away from the sensing anode, being separated from it by the thin interference-rejecting layer and the porous substructure, and speed of response is limited by the reservoir effect of this spacing.
As a further alternative, the enzyme may be placed within the porous substructure and captured by an outer membrane, but this configuration also has the limitations on speed imposed by the multiple layers, and specifically by the thickness of the porous substructure, for now the enzyme is dispersed in this thick layer and is less accessible to its substrate.
As a still further alternative, the enzyme may be placed within and bonded to the porous substructure so that the third outer membrane may be eliminated. Thus, the polarographic substance, hydrogen peroxide, is produced close to the anode and the enzyme is readily accessible to its substrate. This approach, however, requires very sophisticated enzyme immobilization techniques, and presents difficulties in the control of the diffusion of the substrate which determines the range of linearity of the electrode.
Another problem with such a membrane is that if it is too thin it will not have sufficient strength; whereas, if it is too thick, then the all-important speed of measurement is lengthened beyond that tolerable.
That is, for measurement of the unknown in any one sample, time is consumed while the reaction takes place and the potentiometer equilibrates and records the amount of H.sub.2 O.sub.2 produced. Then, before another sample can be tested the potentiometer must go back down to the null point. As is apparent, when a large number of samples are to be analyzed, any reduction in this time period is quite significant.
Accordingly, the need exists for an enzyme electrode membrane which will prevent passage of both high and low molecular weight interfering chemicals, and does not require an inordinant amount of time for sample measurement.